Journal
JOURNAL OF PROTEOME RESEARCH
Volume 16, Issue 8, Pages 3068-3082Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.jproteome.7b00432
Keywords
protein-protein interaction; affinity purification; trypsin digestion; mass spectrometry; selected reaction monitoring
Categories
Funding
- National Institutes of Health [NCATS UL1TR001439, DMS-1361411/DMS-1361318, NIAID AI062885, NIEHS P30 ES006676]
- Sealy Center for Molecular Sciences (SCMM-SysBio)
- Division Of Mathematical Sciences
- Direct For Mathematical & Physical Scien [1361411] Funding Source: National Science Foundation
Ask authors/readers for more resources
Affinity purification-mass spectrometry (AP-MS) has become the method of choice for discovering protein protein interactions (PPIs) under native conditions. The success of AP-MS depends on the efficiency of trypsin digestion and the recovery of the tryptic peptides for MS analysis. Several different protocols have been used for trypsin digestion of protein complexes in AP-MS studies, but no systematic studies have been conducted on the impact of trypsin digestion conditions on the identification of PPIs. Here, we used NF kappa B/RelA and Bromodomain-containing protein 4 (BRD4) as baits and test five distinct trypsin digestion methods (two using on-beads, three using elution-digestion protocols). Although the performance of the trypsin digestion protocols change slightly depending on the different baits, antibodies and cell lines used, we found that elution digestion methods consistently outperformed on-beads digestion methods. The high-abundance interactors can be identified universally by all five methods, but the identification of low-abundance RelA interactors is significantly affected by the choice of trypsin digestion method. We also found that different digestion protocols influence the selected reaction monitoring (SRM)-MS quantification of PPIs, suggesting that optimization of trypsin digestion conditions may be required for robust targeted analysis of PPIs.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available